Promotion of axonal regeneration

ABSTRACT

The present invention concerns a method of promoting axonal regeneration. In particular, the invention concerns a method of promoting the growth or regeneration of neurons, and treating disease or conditions associated with the loss, loss of function or dysfunction of nerve cells, in particular thalamic nerve cells, by administering a polypeptide having a high degree of sequence identity with a native sequence Netrin G1 (NGL-1) or an agonist thereof.

BACKGROUND OF THE INVENTION

1. Field of the Invention

The present invention concerns a method of promoting axonalregeneration. In particular, the invention concerns a method ofpromoting the growth or regeneration of neurons, and treating disease orconditions associated with the loss, loss of function or dysfunction ofnerve cells, in particular thalamic nerve cells, by administering apolypeptide having a high degree of sequence identity with a nativesequence Netrin G1 (NGL-1) or an agonist thereof.

2. Description of the Related Art

The Netrins are a family of laminin-related, diffusible axon guidancemolecules that are conserved from C. elegans to vertebrates [Ishii, N.,Wadsworth, W. G., Stern, B. D., Culotti, J. G. & Hedgecock, E. M.“UNC-6, a laminin-related protein, guides cell and pioneer axonmigrations in C. elegans”. Neuron 9, 873-881. (1992); Serafini, T. etal. “The netrins define a family of axon outgrowth-promoting proteinshomologous to C. elegans UNC-6”, Cell 78, 409-424. (1994)] Thevertebrate netrins are highly expressed in the ventral midline of thecentral nervous system (CNS), attracting the commissural axons andrepelling the trochlear motor axons [Ishii, N., Wadsworth, W. G., Stem,B. D., Culotti, J. G. & Hedgecock, E. M. “UNC-6, a laminin-relatedprotein, guides cell and pioneer axon migrations in C. elegans”. Neuron9, 873-881. (1992)]. Axonal attraction to Netrins is mediated mainly bythe transmembrane receptor DCC, whereas its repulsive action isdependent on both the DCC and Unc5 receptors [Hedgecock, E. M., Culotti,J. G. & Hall, D. H. “The unc-5, unc-6, and unc-40 genes guidecircumferential migrations of pioneer axons and mesodermal cells on theepidermis in C. elegans”, Neuron 4, 61-85. (1990); Hamelin, M., Zhou,Y., Su, M. W., Scott, I. M. & Culotti, J. G. “Expression of the UNC-5guidance receptor in the touch neurons of C. elegans steers their axonsdorsally”, Nature 364, 327-330. (1993); Keino-Masu, K. et al. “Deletedin Colorectal Cancer (DCC) encodes a netrin receptor”, Cell 87, 175-185.(1996); Leonardo, E. D. et al. “Vertebrate homologues of C. elegansUNC-5 are candidate netrin receptors”, Nature 386, 833-838. (1997)].

Recently a Netrin-related molecule, Netrin-G1 (also named as Laminet-1),was identified and shown to be distinguished from the classical netrinsin a number of aspects [Nakashiba, T. et al. “Netrin-G1: a novelglycosyl phosphatidylinositol-linked mammalian netrin that isfunctionally divergent from classical netrins” J. Neurosci. 20,6540-6550. (2000); Yin, Y., Miner, J. H. & Sanes, J. R. “Laminets:laminin- and netrin-related genes expressed in distinct neuronalsubsets”, Mol. Cell. Neurosci. 19, 344-358. (2002)]. See also, WO99/63088, published Dec. 9, 1999, disclosing the sequence of Netrin-G1(originally designated PRO1133, encoded by DNA53913), and WO 01/68848,published Sep. 20, 2001, including microarray data demonstrating theover-expression of Netrin-G1 (PRO1133) in tumor.

Unlike other Netrins, Netrin-G1 is predominantly tethered to the cellmembrane via a C-terminal glycosyl-phosphatidylinositol (GPI) anchor andis not expressed in the ventral midline of the CNS. Instead it is foundin sets of projection neurons such as the mitral cells of the olfactorybulb, the deep cerebellar nuclei and the dorsal thalamus. Multiplesplice variants of Netrin-G1 have been uncovered, suggesting potentialcomplexity of this gene. More importantly, none of the multiple isoformsof Netrin-G1 binds DCC or Unc5, the identified netrin receptors[Nakashiba, T. et al. “Netrin-G1: a novel glycosylphosphatidylinositol-linked mammalian netrin that is functionallydivergent from classical netrins”, J. Neurosci. 20, 6540-6550. (2000)].Therefore its function and mode of action were not known.

The thalamocortical axons (TCAs) project from the dorsal thalamus to thecerebral cortex. These axons first extend toward the ventral thalamusand then turn 90° rostrally, coursing through the ventral telencephalon(i.e. subpallium/the striatum) within the internal capsule and they turndorsally to reach their final target, the cerebral cortex. Along thiscomplex trajectory, both attractive and repulsive signals guide thethalamocortical axons [Braisted, J. E., Tuttle, R. & O'Leary D, D.“Thalamocortical axons are influenced by chemorepellent andchemoattractant activities localized to decision points along theirpath”, Dev. Biol. (Orlando) 208, 430-440 (1999)]. Specifically, therepulsive signals Slit-1 and Slit-2 are required to steer thethalamocortical axons away from the ventral midline region of thediencephalons [Bagri, A. et al. “Slit proteins prevent midline crossingand determine the dorsoventral position of major axonal pathways in themammalian forebrain”, Neuron 33, 233-248. (2002)], while Netrin-1expressed in the ventral telencephalon appear to attract thethalamocortical axons into the internal capsule [Braisted, J. E. et al.“Netrin-1 promotes thalamic axon growth and is required for properdevelopment of the thalamocortical projection,” J. Neurosci 20,5792-5801 (2000)]. However, a substantial population of thethalamocortical axons is still able to reach the internal capsule andthe cerebral cortex in the Netrin1-deficient mice [Bagri, A. et al.“Slit proteins prevent midline crossing and determine the dorsoventralposition of major axonal pathways in the mammalian forebrain.” Neuron33, 233-248. (2002)] suggesting that additional attractive factor(s)must be involved.

A leucine-rich repeat containing polypeptide designated PRO331 (encodedby DNA40981), was disclosed in WO 99/142328, published Mar. 25, 1999 andWO 00/15796, published Mar. 23, 2000, and shown to inhibitVEGF-stimulated proliferation of endothelial cell growth. WO 01/04311,published Jan. 18, 2001 disclosed results demonstrating that the samemolecule has pro-inflammatory properties.

In Example 171 of WO 00/73454, published Dec. 7, 2000, PRO331 was shownto bind PRO1133 (now named Netrin-G1) and vice versa.

SUMMARY OF THE INVENTION

The present invention is, at least part, based on experimental datademonstrating that a human polypeptide, originally designated PRO331,and hereinafter termed Netrin-G1 Ligand, or NGL-1, not only binds to butis a functional ligand of polypeptide PRO1133 (hereafter referred to asNetrin-G1), and that this receptor-ligand pair plays an important rolein neural regeneration and axon outgrowth.

In one aspect, the invention concerns a method of promoting axonalgrowth or regeneration comprising delivering to an injured neuron aneffective amount of a polypeptide having at least about 80% sequenceidentity with amino acid residues 44-352 of SEQ ID NO: 1 and comprisinga transmembrane region, or an agonist thereof.

In particular embodiments, the polypeptide has at least about 85%, or atleast about 90%, or at least about 95%, or at least about 98% or atleast about 99% sequence identity with amino acid residues 44-352 of SEQID NO: 1.

In another embodiment, the polypeptide further comprises a C-terminalPDZ domain-binding motif, where the PDZ domain-binding motif may, forexample, have the sequence of VQETQI (SEQ ID NO: 7).

In yet another embodiment, the polypeptide comprises an extracellulardomain (ECD) comprising nine leucine-rich repeats (LRRs), and mayfurther comprise an N-terminal signal sequence.

In a further embodiment, the polypeptide comprises amino acids 44-352 ofSEQ ID NO: 1.

In a still further embodiment, the polypeptide comprises amino acids44-428 of SEQ ID NO: 1.

In an additional embodiment, the polypeptide comprises amino acids44-546 of SEQ ID NO: 1.

The polypeptide employed in the foregoing method may preferably be humanNGL-1 (SEQ ID NO: 1), with or without the N-terminal signal sequence andwith or without an immunoglobulin-like region, or a non-human homologueof human NGL-1 (SEQ ID NO: 1), with or without an N-terminal signalsequence and with or without an immunoglobulin-like region. In specificembodiments, the non-human homologue can be mouse NGL-1 (SEQ ID NO: 4),or chicken NLG-1 (SEQ ID NO: 5).

The NGL-1 agonist can, for example, be an agonist antibody, includingantibody fragments, or a small molecule, including small organicmolecules and peptides.

In another aspect, the invention concerns a method for treating adisease or condition associated with the loss, loss of function ordysfunction of nerve cells comprising delivering to said nerve cells aneffective amount of a polypeptide having at least 80% sequence identitywith amino acid residues 44-352 of SEQ ID NO: 1 and comprising atransmembrane region, or an agonist thereof.

In a particular embodiment, the nerve cells are thalamic nerve cells.

In another embodiment, the disease or condition is a neurodegenerativedisease.

In a further embodiment, the disease or condition is characterized bynerve cell injury, where the injury may, for example, be due tomechanical trauma, or associated with diabetes, stroke, liver or kidneydysfunction, other endocrine or metabolic derangements, chemotherapy orradiation, or chemical intoxication of the nervous system, or spinalcord injury. Thus, the disease or condition may be allodynia or painfollowing spinal cord injury.

In another embodiment, the disease or condition is associated withneural dysfunction, and can, for example, be selected from the groupconsisting of Alzheimer's disease, Parkinson's disease, Huntington'schorea, amylotrophic lateral sclerosis (ALS), and peripheralneuropathies.

In another embodiment, the disease or condition is a congenital orhereditary abnormality, such as a Charcot-Marie-Tooth disease.

In yet another embodiment, the disease or condition is an autoimmunedisease attacking axons of the central or peripheral nervous system,such as multiple sclerosis or Gulliam-Barre syndrome.

In a further aspect, the invention concerns a method for treating adisease or condition associated with the loss, loss of function ordysfunction of nerve cells comprising delivering to said nerve cells aneffective amount of a nucleic acid encoding a polypeptide having atleast 80% sequence identity with amino acid residues 44-352 of SEQ IDNO: 1 and comprising a transmembrane region, or a peptide or polypeptideagonist thereof. In a preferred embodiment, the nerve cells are thalamicnerve cells. All embodiment discussed above in connection with otheraspects of the invention are also embodiments of the present method, andare within the scope of the invention.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1: Specific and Direct Interaction of NGL-1 and Netrin-G1 on CellSurface

Recombinant human NGL-1-Fc (a-c), human Netrin-G1-Fc (d-f) and humanDCC-Fc (g-i) were used to label COS7 cells expressing human NGL-1 (a, d,g), human Netrin-G1 (b, e, h), chicken Netrin-1 (c, f, i). Specificbinding of the transfected cells was evident for Netrin-G1-expressingcells labeled with NGL-1-Fc (b), for NGL-1-expressing cells labeled withNetrin-G 1-Fc (d), as well as for Netrin-1-expressing cells labeled withDCC-Fc (i). (j) The recombinant ECD-Fc fusion proteins (5 nM) indicatedat the top (lanes 3-7) were incubated with human Netrin-G1-his (firstrow), chicken Netrin-1-his (second row) or human HGF protein (third row)in a solution binding assay. Complexes were pulled down by proteinA-conjugated beads and probed with His-Probe (anti-His), antibodies tochicken netrin-1 and monoclonal antibodies to human HGF, as indicated.Direct and specific binding of NGL-1-Fc and netrin-G1-His (50 kDa) isshown in the first row. The netrin-1-His protein (75-85 kDa) bindsstrongly to DCC-Fc and Unc5-Fc (lanes 3 and 4, second row), extremelyweakly to NGL-1-Fc (lane 5, second row), and not at all to human cMet-Fc(lane 6). HGF binds to cMet-Fc (lane 6, third row). Lane 1, directlyloaded netrin-G1-His; lane 2, directly loaded netrin-1-His; lane 7,directly loaded HGF.

FIG. 2: Characterization of Interaction of NGL-1 and Netrin-G1

(a) Saturation curve and Scatchard analysis of human NGL-1-Fc proteinsolution (concentration in x-axis) to microtiter wells coated with humanNetrin-G1-his protein. The y-axis (Bound) represents specific binding,i.e. total binding minus non-specific binding as determined by cMet-Fcbinding to the wells in parallel. Inset, the Scatchard analysis of thedata gave a Kd=1.6 nM. Linear regression yielded a correlationcoefficient, r=0.96.

(b) A schematic diagram summarizes the predicted structure of theextracellular region of human NGL-1 protein. SS, signal sequence; LRR(leucine-rich repeat); NT, N-terminal domain of LRR; CT, C-terminaldomain of LRR; Ig (immunoglobulin domain) and the ability of thedifferent regions to bind Netrin G1.

FIG. 3: Structure of NGL-1 and Related Molecules

The amino acid sequence alignment of the human NGL-1 (SEQ ID NO: 1) andrelated human proteins encoded by EST cDNAs. NAG14 (GenBank AccessionNumber AF196976; SEQ ID NO: 2) and another related EST, HSM (GenBankAccession Number HSM802162; SEQ ID NO: 3) are closely related to NGL-1.The identical amino acid residues are shaded in black and the conservedresidues in gray. Underlined are the predicted structural domains andmotifs. As shown, NGL-1 includes an N-terminal signal peptide at aminoacid residues 1-42, nine leucine-rich repeats (LRRs) at amino acidresidues, an Ig-like domain at amino acid residues 367-430, atransmembrane region at amino acid residues 563-589, an a C-terminal PDZmotif. Similar motifs for NSG14 and HSM are also shown.

FIG. 4: Sequence Alignment of Human, Mouse and Chicken NGL FamilyMembers

Amino acid sequence alignment of human NGL-1 (SEQ ID NO: 1); mouse NGL(GanBank Accession Number AK032567; SEQ ID NO: 4); chicken NGL (SEQ IDNO: 5); human NAG14 (GenBank Accession Number AF196976; SEQ ID NO: 2)and mouse NAG14 (GenBank Accession Number AF300458; SEQ ID NO: 6). Thechicken NGL protein sequence is derived from the partial cDNA clone byRT-PCR. The identical amino acid residues are shaded in black and theconserved residues in gray.

FIG. 5: Distribution of NGL-1, NetrinG1 Transcripts and the EndogenousNGL-1 Binding Activity

(a) Northern analysis of NGL-1 and Netrin-G1 in adult human tissues andbrain regions as indicated above each panel. (b, c) NGL-1 mRNA in situhybridization of mouse brain at E14, hybridized with digoxigenin-labeledmouse NGL-1 riboprobe. Ctx, cerebral cortex; DT, dorsal thalamus; VT,ventral thalamus; Str, striatum; S, septal area; Ret, retina. (d-h)Endogenous NGL-1 binding activity as labeled by human NGL-1-Fc fusionprotein using vibratome sections (d, e) or whole mount brain tissues (g,h). (g) is the lateral view of the E14 mouse brain, (h) is the ventralview of the E17 mouse brain. The brown color was developed byhorseradish peroxidase (HRP)-conjugated anti-human IgG Fc and HRPsubstrate DAB. The arrows outline the thalamocortical axons in (d, e, g)and lateral olfactory tract in (h). Tel, telencephalon; OB, olfactorybulb. Other abbreviations are the same as in (b, c). (f) When humanNetrin-G1-Fc fusion protein was used for labeling the vibratome sectionsat the similar axial level, the HRP signal was the same as thebackground level assessed by omitting Fc fusion protein in parallelexperiments. Abbreviations are the same as in (b, c). Scale bar, 1 mm(b, c), 1.5 mm (d, e, f), 1.2 mm (g), 4 mm (h).

FIG. 6: Biological Activity of NGL-1

(a-g) Dissociated cultures of E13-E14 mouse thalamic neurons grown oncontrol bovine serum albumin (BSA) substrate (a, b) or on human NGL-1substrate (c-f). Some of the cultures were treated withphosphotidylinositol-specific phospholipase C (PIPLC) (e, f) for 20 minbefore the addition of soluble Netrin-G1 (f) or soluble GFRα3 (g). 44-48hours after the culture, the neurons were stained for the vital dyeCM-FDA (a, c, e, f) or for the neuronal marker, anti-type III β-Tubulin(b, d). The great majority of live cells were positive for neuronalmarker (90-95%) (g). (h-j) Chick thalamofugal axons (equivalent to themammalian thalamocortical axons), whole mount stained by anti-Axonin-1antibody, were significantly reduced when repeated injections of solublehuman NGL-1-Fc protein were given (arrows in i, j). The control PBS-(h)or cMet-Fc (not shown) injected embryos showed normal pattern ofthalamofugal axonal growth. DT, dorsal thalamus; VT, ventral thalamus;Tel, telencephalon. Scale bar, 50 μm (a-f), 0.2 m (h-j).

DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS A. Definitions

Unless defined otherwise, technical and scientific terms used hereinhave the same meaning as commonly understood by one of ordinary skill inthe art to which this invention belongs. See, e.g. Singleton et al.,Dictionary of Microbiology and Molecular Biology 2nd ed., J. Wiley &Sons (New York, N.Y. 1994); Sambrook et al., Molecular Cloning, ALaboratory Manual, Cold Springs Harbor Press (Cold Springs Harbor, N.Y.1989). For purposes of the present invention, the following terms aredefined below.

The terms “PRO331,” “NGL-1,” and “NGL” are used interchangeably andencompass native sequence NGL-1 polypeptides and their functionalderivatives.

A “native sequence NGL-1” or “native NGL-1” comprises a polypeptidehaving the same amino acid sequence as a NGL-1 derived from nature. Thusa native sequence NGL-1 can have the sequence of a human NGL-1 or NGL-1from any other non-human animal, such as vertebrate, mammalian or avianspecies. The term specifically includes native human NGL-1 of SEQ ID NO:1, mouse NGL-1 of SEQ ID NO: 4, chicken NGL-1 of SEQ ID NO: 5, all withor without the N-terminal methionine and with or without the N-terminalsignal sequence. Such native sequence NGL-1 polypeptides can be isolatedfrom nature or produced by recombinant and/or synthetic means. The term“native sequence NGL-1” specifically encompasses naturally occurringtruncated forms (e.g. alternatively spliced forms), and naturallyoccurring allelic variants of the NGL-1. The preferred native sequenceNGL-1 is the mature native sequence human NGL-1 of SEQ ID NO: 1.

A “functional derivative” of a native sequence polypeptide is a compoundhaving a qualitative biological activity in common with the nativesequence polypeptide. For the purpose of the present invention, a“functional derivative” of a native sequence NGL-1 polypeptide isdefined by its ability to bind to and signal through a Netrin-G1receptor. Thus, a “functional derivative” of NGL-1 is a functionalligand of Netrin-G1.

The functional derivatives include amino acid sequence variants(substitution, deletion and/or insertion variants) of a native sequenceNGL-1, which preferably have at least about 60%, or at least about 70%,or at least about 80%, or at least about 85%, or at least about 90%, orat least about 95%, or at least about 98% or at least about 99% overallamino acid sequence identity with a native sequence NGL-1 polypeptide,such as a human NGL-1 (SEQ ID NO: 1); mouse NGL (SEQ ID NO: 4), orchicken NGL (SEQ ID NO: 5). Fragments of native sequence NGL-1polypeptides are specifically included within the definition offunctional derivatives, provided that they include at least thesequences of the corresponding native sequence NGL-1 that are requiredfor binding to and signaling through a native Netrin-G1 receptor. Sincesoluble NGL-1 is capable of binding netrin-G1, but is not sufficient toactivate the downstream signaling pathway, the functional derivativespreferably are membrane-bound proteins. Structure-function studies haveshown that the Ig-like region within SEQ ID NO: 1 is not required fornetrin-G1 binding, and that the last six C-terminal amino acids may beneeded for activity. Accordingly, NGL-1 variants preferably retain theseC-terminal amino acids but may lack all or part of the Ig-like region.

A specific group of NGL-1 functional derivatives includes NGL-1 aminoacid sequence variants encoded by nucleic acid hybridizing understringent conditions to the complement of nucleic acid encoding a nativeNGL-1 polypeptide.

Amino acid sequence variants of NGL-1 specifically include a polypeptidehaving at least 80%, or at least 85%, or at least 90%, or at least 95%,or at least 98%, or at least 99% sequence identity with amino acidresidues 44-352 of SEQ ID NO: 1 and comprising a transmembrane region.Such amino acid sequence variants may further comprise a C-terminal PDZdomain-binding motif, such as a PDZ-binding motif having the sequence ofVQETQI (SEQ ID NO: 7). Other amino acid variants comprise theextracellular domain (ECD) of a native sequence NGL-1, comprising nineleurine-rich repeats (LRRs). NGL-1 variants comprising amino acids44-352, or 44-546 of SEQ ID NO: 1 are specifically included. Furtheramino acid sequence variants comprise amino acid residues 43-365, or43-653 of human NAG14 (AF196976) of SEQ ID NO: 2. Yet another group ofamino acid sequence variants include amino acid residues 1-147 of HSM(HSM802162) of SEQ ID NO: 3.

Functional derivatives further include fusion polypeptides including anative sequence or variant NGL-1 polypeptide (e.g. a fragment, such asthe extracellular domain of a native sequence NGL-1) fused to aheterologous sequence, such as a tag or an immunoglobulin sequence aswell as glycosylation variants of native sequence and variant NGL-1polypeptides.

Glycosylation variants are NGL-1 polypeptides and polypeptide variantsthat differ in the extent of their glycosylation and/or in theirglycosylation patterns. Glycosylation variants include polypeptidescompletely lacking in glycosylation (unglycosylated), variants havingless glycosylated sites than the native form (deglycosylated) as well asvariants in which the glycosylation has been changed. Included aredeglycosylated and unglycosylated amino acid sequences variants,deglycosylated and unglycosylated native sequence NGL-1 molecules, andother glycosylation variants. For example, substitutional or deletionalmutagenesis may be employed to eliminate the N- or O-linkedglycosylation sites in the native sequence or variant NGL-1 molecule,e.g. the asparagine residue may be deleted or substituted for anotherbasic residue such as lysine or histidine. Alternatively, flankingresidues making up the glycosylation site may be substituted or deleted,even though the asparagine residues remain unchanged, in order toprevent glycosylation by eliminating the glycosylation recognition site.Additionally, unglycosylated NGL-1 polypeptides which have theglycosylation sites of a native molecule may be produced in recombinantprokaryotic cell culture because prokaryotes are incapable ofintroducing glycosylation into polypeptides.

Glycosylation variants may be produced by selecting appropriate hostcells or by in vitro methods. Yeast and insect cells, for example,introduce glycosylation which varies significantly from that ofmammalian systems. Similarly, mammalian cells having a different speciesor tissue origin than the source of the NGL-1 polypeptide are routinelyscreened for the ability to introduce variant glycosylation ascharacterized for example by elevated levels of mannose or variantratios of mannose, fucose, sialic acid, and other sugars typically foundin mammalian glycoproteins.

The term “Netrin-G1” encompasses native sequence Netrin-G1 and itsfunctional derivatives. Functional derivatives are defined as discussedabove for NGL-1, and include

“Native sequence Netrin-G1” or “native Netrin-G1” comprises apolypeptide having the same amino acid sequence as a Netrin-G1 derivedfrom nature. Thus a native sequence Netrin-G1 can have the sequence of ahuman Netrin-G1 or Netrin-G1 from any other non-human animal, such asvertebrate, mammalian or avian species. Native sequence Netrin-G1 iswell known in the art, see, for example, Nakashiba, T. et al.,“Netrin-G1: a novel glycosyl phosphatidylinositol-linked mammaliannetrin that is functionally divergent from classical netrins”, J.Neurosci 20 6540-6550 (2000) and Yin et al., “Laminets: laminin- andnetrin-related genes expressed in distinct neuronal subsets”, Mol. Cell.Neurosci 1'9 344-358 (2002) which are expressly incorporated byreference herein. Such native sequence Netrin-G1 polypeptides can beisolated from nature or produced by recombinant and/or synthetic means.The term “native sequence Netrin-G1” specifically encompasses naturallyoccurring truncated forms of the Netrin-G1, naturally occurring allelicvariants of the Netrin-G1. The preferred native sequence Netrin-G1 is amature native sequence of human Netrin-G1 of SEQ ID NO: 8.

Just as in the case of NGL-1, functional derivatives include amino acidsequence variants, fusion polypeptides, and glycosylation variants.

Preferred amino acid sequence variants of Netrin-G1 have at least about60%, or at least about 70%, or at least about 80%, or at least about85%, or at least about 90%, or at least about 95%, or at least about 98%or at least about 99% overall amino acid sequence identity with a nativesequence Netrin-G1 polypeptide, such as the human Netrin-G1 polypeptideof SEQ ID NO: 8, as long as they retain a qualitative biologicalactivity in common with a native sequence Netrin-G1 polypeptide. Apreferred biological activity is the ability to bind to and mediate thebiological activity of a native sequence NGL-1.

The term “immunoadhesin” and refers to a chimeric molecule that combinesa portion of a ligand or receptor, e.g. NGL-1 or Netrin-G1 (generallythe extracellular domain thereof) with an immunoglobulin sequence. Theimmunoglobulin sequence preferably, but not necessarily, is animmunoglobulin constant domain sequence. The immunoglobulin moiety inthe chimeras of the present invention may be obtained from IgG1, IgG2,IgG3 or IgG4 subtypes, IgA, IgE, IgD or IgM, but preferably IgG1 orIgG3. Thus, for example, the Ig portion of an IgG1 immunoadhesin maycomprise the CH1, hinge, CH2 and CH3 sequences, or hinge, CH2 and CH3sequences of the immunoglobulin constant region.

The term “epitope-tagged” when used herein refers to a chimericpolypeptide comprising a polypeptide, such as NGL-1 or Netrin-G1, fusedto a “tag polypeptide”. The tag polypeptide has enough residues toprovide an epitope against which an antibody thereagainst can be made,yet is short enough such that it does not interfere with biologicalactivity of the NGL-1 or Netrin-G1. The tag polypeptide preferably alsois fairly unique so that the antibody raised against it does notsubstantially cross-react with other epitopes. Suitable tag polypeptidesgenerally have at least six amino acid residues and usually betweenabout 8-50 amino acid residues (preferably between about 9-30 residues).Preferred are poly-histidine sequences, which bind nickel or othertransient metals, allowing isolation of the tagged protein by Ni-NTA orother transient metal chromatography as described (Lindsay et al. Neuron17:571-574 (1996)), for example.

“Isolated NGL-1.” or “isolated Netrin-G1” means material that has beenpurified from a natural source or has been prepared by recombinant orsynthetic methods and is sufficiently free of other peptides or proteins(1) to obtain at least 15 and preferably 20 amino acid residues of theN-terminal or of an internal amino acid sequence by using a spinning cupsequential or the best commercially available amino acid sequentialmarketed or as modified by published methods as of the filing date ofthis application, or (2) to homogeneity by SDS-PAGE under non-reducingor reducing conditions using Coomassie blue or, preferably, silverstain. Homogeneity here means less than about 5% contamination withother source proteins

“Essentially pure” protein means a composition comprising at least about90% by weight of the protein, based on total weight of the composition,preferably at least about 95% by weight. “Essentially homogeneous”protein means a composition comprising at least about 99% by weight ofprotein, based on total weight of the composition.

“Percent amino acid sequence identity” is defined herein as thepercentage of amino acid residues in the candidate sequences that areidentical with the residues in the Netrin-G1 or NGL-1. sequence, afteraligning the sequences and introducing gaps, if necessary, to achievethe maximum percent sequence identity, and not considering anyconservative substitutions as part of the sequence identity. Percentamino acid sequence identity is calculated for the full-length of theNetrin-G1 or NGL-1 sequence. Thus, shorter sequences, even if they show100% sequence identity with a portion of the Netrin-G1 of NGL-1 sequencewill be less than 100% identical with those sequences.

The term “agonist” is used herein in the broadest sense. An “NGL-1agonist” is a molecule which partially or fully mimics a biologicalactivity of a native sequence NGL-1 polypeptide. NGL-1 agonists include,without limitation, agonist antibodies specifically binding NGL-1,peptides, small inorganic molecules, and the like.

The term “antagonist” is used herein in the broadest sense. An NGL-1“antagonist” is a molecule, which partially or fully bocks, inhibits,neutralizes, prevents or interferes with a biological activity of NGL-1,regardless of the underlying mechanism. For the purpose of the presentinvention, the biological activity preferably is the ability toAntagonists of NGL-1 can be identified, for example, based upon theirability to inhibit, block, or reverse binding of NGL-1 to Netrin-G1. Forexample a culture of activated TCA (thalamocortical axons) cells can beincubated with Netrin G1, in the presence and absence of a testcompound, and NGL-1 binding monitored in the cell culture. If the NGL-1binding even is lower in the presence of the test compound than in itsabsence, the test compound is an NGL-1 antagonist. Furthermore, NGL-1antagonists can be identified in functional assays, by measuring NGL-1biological activity. Examples of NGL-1 antagonists include, withoutlimitation, neutralizing antibodies against the native sequence NGL-1polypeptide, immunoadhesins comprising a soluble NGL-1 fused to animmunoglobulin constant region sequence, small molecules, antisenseoligonucleotides capable of inhibiting translation and/or transcriptionof a gene encoding a NGL-1 polypeptide, oligonucleotide decoys, etc.

“Antisense oligodeoxynucleotides” or “antisense oligonucleotides” (whichterms are used interchangeably) are defined as nucleic acid moleculesthat can inhibit the transcription and/or translation of target genes ina sequence-specific manner. The term “antisense” refers to the fact thatthe nucleic acid is complementary to the coding (“sense”) geneticsequence of the target gene. Antisense oligonucleotides hybridize in anantiparallel orientation to nascent mRNA through Watson-Crickbase-pairing. By binding the target mRNA template, antisenseoligonucleotides block the successful translation of the encodedprotein. The term specifically includes antisense agents called“ribozymes” that have been designed to induce catalytic cleavage of atarget RNA by addition of a sequence that has natural self-splicingactivity (Warzocha and Wotowiec, “Antisense strategy: biological utilityand prospects in the treatment of hematological malignancies.” Leuk.Lymphoma 24:267-281 [1997]).

“Oligonucleotide decoy” molecules, also referred to a transcriptionfactor oligonucleotide decoys (TF ODNs) are small double-strandedoligonucleotides that are introduced into cells to specifically bind totarget transcription factors, thereby, preventing these factors fromtransactivating their target genes.

“Native antibodies and immunoglobulins” are usually heterotetramericglycoproteins of about 150,000 daltons, composed of two identical light(L) chains and two identical heavy (H) chains. Each light chain islinked to a heavy chain by one covalent disulfide bond, while the numberof disulfide linkages varies between the heavy chains of differentimmunoglobulin isotypes. Each heavy and light chain also has regularlyspaced intrachain disulfide bridges. Each heavy chain has at one end avariable domain (V_(H)) followed by a number of constant domains. Eachlight chain has a variable domain at one and (V_(L)) and a constantdomain at its other end; the constant domain of the light chain isaligned with the first constant domain of the heavy chain, and the lightchain variable domain is aligned with the variable domain of the heavychain. Particular amino acid residues are believed to form an interfacebetween the light and heavy chain variable domains (Clothia et al., J.Mol. Biol., 186:651-663 (1985); Novotny and Haber, Proc. Natl. Acad.Sci. USA, 82:4592-4596 (1985)).

The term “variable” refers to the fact that certain portions of thevariable domains differ extensively in sequence among antibodies and areused in the binding and specificity of each particular antibody for itsparticular antigen. However, the variability is not evenly distributedthrough the variable domains of antibodies. It is concentrated in threesegments called complementarity determining regions (CDRs) orhypervariable regions both in the light chain and the heavy chainvariable domains. The more highly conserved portions of variable domainsare called the framework (FR). The variable domains of native heavy andlight chains each comprise four FR regions, largely adopting a b-sheetconfiguration, connected by three CDRs, which form loops connecting, andin some cases forming part of, the b-sheet structure. The CDRs in eachchain are held together in close proximity by the FR regions and, withthe CDRs from the other chain, contribute to the formation of theantigen binding site of antibodies (see Kabat et al., Sequences ofProteins of Immunological Interest, National Institute of Health,Bethesda, Md. (1987)). The constant domains are not involved directly inbinding an antibody to an antigen, but exhibit various effectorfunctions, such as participation of the antibody in antibody-dependentcellular toxicity.

Papain digestion of antibodies products two identical antigen bindingfragments, called “Fab” fragments, each with a single antigen bindingsite, and a residual “Fc” fragment, whose name reflects its ability tocrystallize readily. Pepsin treatment yields an F(ab′).sub.2 fragmentthat has two antigen combining sites and is still capable ofcross-linking antigen.

“Fv” is the minimum antibody fragment which contains a complete antigenrecognition and binding site. This region consists of a dimer of oneheavy and one light chain variable domain in tight, non-covalentassociation. It is in this configuration that the three CDRs of eachvariable domain interact to define an antigen binding site on thesurface of the V_(H)-V_(L) dimer. Collectively, the six CDRs conferantigen binding specificity to the antibody. However, even a singlevariable domain (or half of an Fv comprising only three CDRs specificfor an antigen) has the ability to recognize and bind antigen, althoughat a lower affinity than the entire binding site.

The Fab fragment also contains the constant domain of the light chainand the first constant domain (CH1) of the heavy chain. Fab″ fragmentsdiffer from Fab fragments by the addition of a few residues at thecarboxy terminus of the heavy chain CH1 domain including one or morecysteines from the antibody hinge region. Fab′-SH is the designationherein for Fab′ in which the cysteine residue(s) of the constant domainsbear a free thiol group. F(ab′)₂ antibody fragments originally wereproduced as pairs of Fab′ fragments which have hinge cysteines betweenthem. Other, chemical couplings of antibody fragments are also known.

The “light chains” of antibodies (immunoglobulins) from any vertebratespecies can be assigned to one of two clearly distinct types, calledkappa and lambda (1), based on the amino acid sequences of theirconstant domains.

Depending on the amino acid sequence of the constant domain of theirheavy chains, immunoglobulins can be assigned to different classes.There are five major classes of immunoglobulins: IgA, IgD, IgE, IgG andIgM, and several of these may be further divided into subclasses(isotypes), e.g., IgG-1, IgG-2, IgG-3, and IgG-4; IgA-1 and IgA-2. Theheavy chain constant domains that correspond to the different classes ofimmunoglobulins are called alpha, delta, epsilon, gamma and .mu.,respectively. The subunit structures and three-dimensionalconfigurations of different classes of immunoglobulins are well known.

The term “antibody” is used in the broadest sense and specificallycovers single monoclonal antibodies (including agonist and antagonistantibodies), antibody compositions with polyepitopic specificity, aswell as antibody fragments (e.g., Fab, F(ab′)₂, scFv and Fv), so long asthey exhibit the desired biological activity.

The term “monoclonal antibody” as used herein refers to an antibodyobtained from a population of substantially homogeneous antibodies,i.e., the individual antibodies comprising the population are identicalexcept for possible naturally occurring mutations that may be present inminor amounts. Monoclonal antibodies are highly specific, being directedagainst a single antigenic site. Furthermore, in contrast toconventional (polyclonal) antibody preparations which typically includedifferent antibodies directed against different determinants (epitopes),each monoclonal antibody is directed against a single determinant on theantigen. In addition to their specificity, the monoclonal antibodies areadvantageous in that they are synthesized by the hybridoma culture,uncontaminated by other immunoglobulins. The modifier “monoclonal”indicates the character of the antibody as being obtained from asubstantially homogeneous population of antibodies, and is not to beconstrued as requiring production of the antibody by any particularmethod. For example, the monoclonal antibodies to be used in accordancewith the present invention may be made by the hybridoma method firstdescribed by Kohler & Milstein, Nature, 256:495 (1975), or may be madeby recombinant DNA methods (see, e.g., U.S. Pat. No. 4,816,567 (Cabillyet al.)).

The monoclonal antibodies herein specifically include “chimeric”antibodies (immunoglobulins) in which a portion of the heavy and/orlight chain is identical with or homologous to corresponding sequencesin antibodies derived from a particular species or belonging to aparticular antibody class or subclass, while the remainder of thechain(s) is identical with or homologous to corresponding sequences inantibodies derived from another species or belonging to another antibodyclass or subclass, as well as fragments of such antibodies, so long asthey exhibit the desired biological activity, e.g. binding to andactivating mpl (U.S. Pat. No. 4,816,567 (Cabilly et al.); and Morrisonet al., Proc. Natl. Acad. Sci. USA, 81:6851-6855 (1984)).

“Humanized” forms of non-human (e.g., murine) antibodies are chimericimmunoglobulins, immunoglobulin chains or fragments thereof (such as Fv,Fab, Fab′, F(ab′)₂ or other antigen-binding subsequences of antibodies)which contain minimal sequence derived from non-human immunoglobulin.For the most part, humanized antibodies are human immunoglobulins(recipient antibody) in which residues from a complementary determiningregion (CDR) of the recipient are replaced by residues from a CDR of anon-human species (donor antibody) such as mouse, rat or rabbit havingthe desired specificity, affinity and capacity. In some instances, Fvframework residues of the human immunoglobulin are replaced bycorresponding non-human residues. Furthermore, humanized antibody maycomprise residues which are found neither in the recipient antibody norin the imported CDR or framework sequences. These modifications are madeto further refine and optimize antibody performance. In general, thehumanized antibody will comprise substantially all of at least one, andtypically two, variable domains in which all or substantially all of theCDR regions correspond to those of a non-human immunoglobulin and all orsubstantially all of the FR regions are those of a human immunoglobulinconsensus sequence. The humanized antibody optimally also will compriseat least a portion of an immunoglobulin constant region (Fc), typicallythat of a human immunoglobulin. For further details see: Jones et al.,Nature, 321:522-525 (1986); Reichmann et al., Nature, 332:323-329(1988); and Presta, Curr. Op. Struct. Biol., 2:593-596 (1992)).

“Single-chain Fv” or “sFv” antibody fragments comprise the V_(H) andV_(L) domains of antibody, wherein these domains are present in a singlepolypeptide chain. Generally, the Fv polypeptide further comprises apolypeptide linker between the V_(H) and V_(L) domains which enables thesFv to form the desired structure for antigen binding. For a review ofsFv see Pluckthun in The Pharmacology of Monoclonal Antibodies, vol.113, Rosenburg and Moore eds. Springer-Verlag, New York, pp. 269-315(1994).

The term “diabodies” refers to small antibody fragments with twoantigen-binding sites, which fragments comprise a heavy chain variabledomain (V_(H)) connected to a light chain variable domain (V_(L)) in thesame polypeptide chain (V_(H) and V_(L)). By using a linker that is tooshort to allow pairing between the two domains on the same chain, thedomains are forced to pair with the complementary domains of anotherchain and create two antigen-binding sites. Diabodies are described morefully in, for example, EP 404,097; WO 93/11161; and Hollinger et al.,Proc. Natl. Acad. Sci. USA 90:6444-6448 (1993).

The expression “linear antibodies” when used throughout this applicationrefers to the antibodies described in Zapata et al. Protein Eng. 8(10):1057-1062 (1995). Briefly, these antibodies comprise a pair of tandem Fdsegments (V_(H)-C_(H) 1-V_(H)-C_(H) 1) which form a pair of antigenbinding regions. Linear antibodies can be bispecific or monospecific.

The terms “treat” or “treatment” refer to both therapeutic treatment andprophylactic or preventative measures, wherein the object is to preventor slow down (lessen) an undesired physiological change or disorder. Forpurposes of this invention, beneficial or desired clinical resultsinclude, but are not limited to, alleviation of symptoms, diminishmentof extent of disease, stabilized (i.e., not worsening) state of disease,delay or slowing of disease progression, amelioration or palliation ofthe disease state, and remission (whether partial or total), whetherdetectable or undetectable. “Treatment” can also mean prolongingsurvival as compared to expected survival if not receiving treatment.Those in need of treatment include those already with the condition ordisorder as well as those prone to have the condition or disorder orthose in which the condition or disorder is to be prevented.

“Chronic” administration refers to administration of the agent(s) in acontinuous mode as opposed to an acute mode, so as to maintain thedesired effect for an extended period of time.

“Intermittent” administration is treatment that is not consecutivelydone without interruption, but rather is cyclic in nature.

Administration “in combination with” one or more further therapeuticagents includes simultaneous (concurrent) and consecutive administrationin any order.

A “subject” is a vertebrate, preferably a mammal, more preferably ahuman.

The term “mammal” is used herein to refer to any animal classified as amammal, including, without limitation, humans, non-human primates,domestic and farm animals, and zoo, sports, or pet animals, such assheep, horses, cows, pigs, cats, dogs, etc. Preferably, the mammalherein is human.

An “effective amount” is an amount sufficient to effect beneficial ordesired therapeutic (including preventative) results. An effectiveamount can be administered in one or more administrations.

B. Modes of Carrying Out the Invention

The practice of the present invention will employ, unless otherwiseindicated, conventional techniques of molecular biology (includingrecombinant techniques), microbiology, cell biology, biochemistry andimmunology, which are within the skill of the art. Such techniques areexplained fully in the literature, such as, “Molecular Cloning: ALaboratory Manual”, 2^(nd) edition (Sambrook et al., 1989);“Oligonucleotide Synthesis” (M. J. Gait, ed., 1984); “Animal CellCulture” (R. I. Freshney, ed., 1987); “Methods in Enzymology” (AcademicPress, Inc.); “Handbook of Experimental Immunology”, 4^(th) edition (D.M. Weir & C. C. Blackwell, eds., Blackwell Science Inc., 1987); “GeneTransfer Vectors for Mammalian Cells” (J. M. Miller & M. P. Calos, eds.,1987); “Current Protocols in Molecular Biology” (F. M. Ausubel et al.,eds., 1987); “PCR: The Polymerase Chain Reaction”, (Mullis et al., eds.,1994); and “Current Protocols in Immunology” (J. E. Coligan et al.,eds., 1991).

The present invention, at least in part, is based on the identificationof the interaction between the transmembrane ligand NGL-1 and Netrin-G1as essential for the growth of thalamocortical axons.

NGL-1 Native human NGL-1 was originally disclosed as “PRO331” encoded byDNA40981-1234 in U.S. provisional application Ser. Nos. 60/065,186 filedon Nov. 12, 1997, 60/066,770 filed on Nov. 24, 1997 and 60/088,026 filedon Jun. 4, 1998, and in PCT application publication No. WO 99/14328,published on Mar. 25, 1999. DNA40981 was deposited with the AmericanType Culture Collection (ATCC), Manassas, Va., USA, on Nov. 7, 1997, andassigned ATCC Deposit No. 209439. NGL-1 has been shown to inhibitVEGF-stimulated proliferation of endothelial cell growth (WO 99/14328,published Mar. 25, 1999 and WO 00/14796, published Mar. 23, 2000),exhibited prolinflammatory activity in a skin vascular permeabilityassay (WO 01/04311, published Jan. 18, 2001), and stimulated immuneresponse in the mixed lymphocyte reaction (MLR) assay (WO 01/19991,published Mar. 22, 2001). The human NGL-1 cDNA encodes a type Itransmembrane protein with an N-terminal signal sequence, a singletransmembrane domain of 640 amino acid residues and a predictedmolecular weight of 71.9 kDa (FIG. 2 b and FIG. 3, SEQ ID NO: 1). TheECD of NGL-1 consists of 9 leucine rich repeats (LRR) with the flankingLRR N-terminal domain (LRR-NT) and LRR C-terminal domain (LRR-CT),followed by an immunoglobulin domain (Ig domain). The 92 amino acidcytoplasmic region does not contain any obvious structural consensussequence except that the C-terminus sequence “ETQI” (SEQ ID NO: 8) is apotential PDZ-domain binding motif.

Native human Netrin-G1 was originally disclosed as “PRO1133” encoded byDNA53913-1490 in U.S. provisional application Ser. No. 60/097,952 filedon Aug. 26, 1998. DNA53913-1490 was deposited with ATCC, Manassas, Va.,USA, on Aug. 25, 1998, and assigned ATCC Deposit No. 203162. See also,Nakashiba et al., J. Neurosci. 20:6540-6550 (2000) and Yin et al., Dev.Biol. 208:430-440 (1999). Several splice variants of Netrin-G1 have beendetected, and it was reported that none of the multiple isoforms ofnetrin-G1 binds DCC or Unc5, the known netrin receptors (Nakashiba etal., supra).

Netrin-G1 and NGL-1 were identified as a receptor-ligand pair in Example171 of PCT application Publication No. 00/73454, published on Dec. 7,2000. The same information is present in PCT application Publication No.01/68848, published on Sep. 20, 2001. In particular, a novelhigh-through-put protein interaction assay was utilized to identifynative sequence human NGL-1, a novel LRR- and Ig domain-containingtransmembrane protein, as a specific binding partner of native sequencehuman Netrin-G1, as described in the examples below.

The present invention demonstrates, for the first time, the role of theNetrin-G1/NGL-1 receptor/ligand pair in promoting the outgrowth ofthalamic axons.

Variants of native sequence NGL-1 and Netrin-G1 can be prepared bymethods known in the art. Thus, amino acid sequence variants arepolypeptides having an amino acid sequence which differs from a nativesequence by virtue of the insertion, deletion, and/or substitution ofone or more amino acid residues within a native sequence. Amino acidsequence variants may be prepared synthetically, such as by introducingappropriate nucleotide changes into a previously isolated NGL-1 orNetrin-G1 DNA, or by in vitro synthesis of the desired variantpolypeptide. As indicated above, such variants will comprise deletionsfrom, or insertions or substitutions of, one or more amino acid residueswithin the amino acid sequence of a native NGL-1 or Netrin-G1polypeptide. Any combination of deletion, insertion, and substitution ismade to arrive at a desired amino acid sequence variant, provided thatthe resulting variant polypeptide possesses a desired characteristic.The amino acid changes also may result in further modifications of NGL-1or Netrin-G1 upon expression in recombinant hosts, e.g. introducing ormoving sites of glycosylation, or introducing membrane anchor sequences(in accordance with PCT WO 89/01041 published Feb. 9, 1989).

There are two principal variables to consider in making suchpredetermined mutations: the location of the mutation site and thenature of the mutation. In general, the location and nature of themutation chosen will depend upon the characteristic to be modified. Forexample, candidate NGL-1 antagonists or super agonists initially will beselected by locating amino acid residues that are identical or highlyconserved among NGL-1 and related native polypeptides, such as, forexample, NAG14 or HSM. Those residues then will be modified in series,e.g., by (1) substituting first with conservative choices and then withmore radical selections depending upon the results achieved, (2)deleting the target, residue, or (3) inserting residues of the same ordifferent class adjacent to the located site, or combinations of options1-3.

One helpful technique is called “ala scanning”. Here, an amino acidresidue or group of target residues are identified and substituted byalanine or polyalanine. Those domains demonstrating functionalsensitivity to the alanine substitutions then are refined by introducingfurther or other variants at or for the sites of alanine substitution.While the site for introducing an amino acid sequence variation ispredetermined, the nature of the mutation per se need not bepredetermined. For example, to optimize the performance of a mutation ata given site, ala scanning or random mutagenesis is conducted at thetarget codon or region and the expressed NGL-1 variants are screened forthe optimal combination of desired activity.

Amino acid sequence deletions generally range from about 1 to 30residues, more preferably about 1 to 10 residues, and typically arecontiguous. Deletions may be introduced into regions of low homologyamong NGL-1 and related native polypeptides, such as NAG14 or HSM tomodify the activity of NGL-1. Deletions from NGL-1 in areas ofsubstantial homology with structurally related polypeptides will be morelikely to modify the biological activity of NGL-1 more significantly.The number of consecutive deletions will be selected so as to preservethe tertiary structure of NGL-1 in the affected domain, e.g.,beta-pleated sheet or alpha helix.

Amino acid sequence insertions include amino- and/or carboxyl-terminalfusions ranging in length from one residue to polypeptides containing athousand or more residues, as well as intrasequence insertions of singleor multiple amino acid residues. Intrasequence insertions (i.e.,insertions within the mature NGL-1 sequence) may range generally fromabout 1 to 10 residues, more preferably 1 to 5, most preferably 1 to 3.An example of a terminal insertion includes fusion of a heterologousN-terminal signal sequence to the N-terminus of the NGL-1 molecule tofacilitate the secretion of mature NGL-1 from recombinant hosts. Suchsignals generally will be homologous to the intended host cell andinclude STII or Ipp for E. coli, alpha factor for yeast, and viralsignals such as herpes gD for mammalian cells. Other insertions includethe fusion of an immunogenic polypeptide such as a bacterial or yeastprotein to the N- or C-termini of NGL-1.

The third group of variants are those in which at least one amino acidresidue in NGL-1, and preferably only one, has been removed and adifferent residue inserted in its place. An example is the replacementof arginine and lysine by other amino acids to render the NGL-1resistant to proteolysis by serine proteases, thereby creating a variantof NGL-1 that is more stable. The sites of greatest interest forsubstitutional mutagenesis include sites where the amino acids found inNGL-1 and structurally related polypeptides are substantially differentin terms of side chain bulk, charge of hydrophobicity, but where therealso is a high degree of homology at the selected site within variousanimal analogues of native human NGL-1.

Amino acid sequence variants of native sequence Netrin-G1 can bedesigned and made in a similar manner.

1. Screening Assays to Identify NGL-1 Agonists

This invention includes the use of NGL-1 and its agonists, and screeningassays to identify NGL-1 agonists.

Screening assays for agonist drug candidates may be designed to identifycompounds that bind or complex with Netrin-G1 (including a subunit orother fragment thereof) and mimic a qualitative biological activity ofnative NGL-1, or otherwise enhance the interaction of Netrin-G1 withNGL-1, thereby enhancing NGL-1 biological activity, such as theproduction and/or functioning of neurons. The screening assays providedherein include assays amenable to high-throughput screening of chemicallibraries, making them particularly suitable for identifying smallmolecule drug candidates. Generally, binding assays and activity assaysare provided.

The assays can be performed in a variety of formats, including, withoutlimitation, protein-protein binding assays (including competitivebinding assays), biochemical screening assays, immunoassays, andcell-based assays, which are well characterized in the art.

All assays for agonists are common in that they call for contacting thedrug candidate with a native sequence Netrin-G1 polypeptide, or afragment of such polypeptide, under conditions and for a time sufficientto allow these two components to interact, and measuring an NGL-1biological activity.

In binding assays, the interaction is binding, and the complex formedcan be isolated or detected in the reaction mixture. In a particularembodiment, either the Netrin G1 or NGL-1 polypeptide or the drugcandidate is immobilized on a solid phase, e.g., on a microtiter plate,by covalent or non-covalent attachments. Non-covalent attachmentgenerally is accomplished by coating the solid surface with a solutionof the Netrin-G1 or NGL-1 polypeptide and drying. Alternatively, animmobilized antibody, e.g., a monoclonal antibody, specific for theNetrin-G1 polypeptide or the NGL-1 polypeptide to be immobilized can beused to anchor it to a solid surface. The assay is performed by addingthe non-immobilized component, which may be labeled by a detectablelabel, to the immobilized component, e.g., the coated surface containingthe anchored component. When the reaction is complete, the non-reactedcomponents are removed, e.g., by washing, and complexes anchored on thesolid surface are detected. When the originally non-immobilizedcomponent carries a detectable label, the detection of label immobilizedon the surface indicates that complexing occurred. Where the originallynon-immobilized component does not carry a label, complexing can bedetected, for example, by using a labeled antibody specifically bindingthe immobilized complex.

If the candidate compound is a polypeptide which interacts with but doesnot bind to Netrin-G1 or the NGL-1 receptor, its interaction with therespective polypeptide can be assayed by methods well known fordetecting protein-protein interactions. Such assays include traditionalapproaches, such as, e.g., cross-linking, co-immunoprecipitation, andco-purification through gradients or chromatographic columns. Inaddition, protein-protein interactions can be monitored by using ayeast-based genetic system described by Fields and co-workers (Fieldsand Song, Nature (London), 340:245-246 (1989); Chien et al., Proc. Natl.Acad. Sci. USA, 88:9578-9582 (1991)) as disclosed by Chevray andNathans, Proc. Natl. Acad. Sci. USA, 89: 5789-5793 (1991). Manytranscriptional activators, such as yeast GAL4, consist of twophysically discrete modular domains, one acting as the DNA-bindingdomain, the other one functioning as the transcription-activationdomain. The yeast expression system described in the foregoingpublications (generally referred to as the “two-hybrid system”) takesadvantage of this property, and employs two hybrid proteins, one inwhich the target protein is fused to the DNA-binding domain of GAL4, andanother, in which candidate activating proteins are fused to theactivation domain. The expression of a GAL1-lacZ reporter gene undercontrol of a GAL4-activated promoter depends on reconstitution of GAL4activity via protein-protein interaction. Colonies containinginteracting polypeptides are detected with a chromogenic substrate forβ-galactosidase. A complete kit (MATCHMAKER™) for identifyingprotein-protein interactions between two specific proteins using thetwo-hybrid technique is commercially available from Clontech. Thissystem can also be extended to map protein domains involved in specificprotein interactions as well as to pinpoint amino acid residues that arecrucial for these interactions.

The NGL-1 agonists can be identified, for example, based upon theirability to enhance axon growth, especially the outgrowth of thalamic,such as thalamocortical axons.

A special group of NGL-1 agonists includes amino acid sequences variantsof native sequence NGL-1 molecules.

Another special group of NGL-1 agonists includes agonist antibodies,such as, for example, agonist anti-NGL-1 antibodies. By “agonistantibody” is meant an antibody which is able to bind to a nativesequence NGL-1 polypeptide, and signal through a Netrin-G1 receptor invitro and/or in vivo. Preferred agonist antibodies herein have theability to promote the outgrowth of thalamocortical axons.

2. Anti-NGL-1 Antibodies

As discussed above, in a particular embodiment, the NGL-1 agonists aremonoclonal antibodies to NGL-1 (e.g. a subunit of NGL-1), includingantibody fragments. Antibodies to the ECD of NGL-1 are specificallywithin the scope of the invention.

Methods for making monoclonal antibodies are well known in the art.Thus, monoclonal antibodies may be prepared using hybridoma methods,such as those described by Kohler and Milstein, Nature, 256:495 (1975).In a hybridoma method, a mouse, hamster, or other appropriate hostanimal, is typically immunized with an immunizing agent to elicitlymphocytes that produce or are capable of producing antibodies thatwill specifically bind to the immunizing agent. Alternatively, thelymphocytes may be immunized in vitro.

The immunizing agent will typically include the NGL-1 polypeptide or afusion protein thereof. Generally, either peripheral blood lymphocytes(“PBLs”) are used if cells of human origin are desired, or spleen cellsor lymph node cells are used if non-human mammalian sources are desired.The lymphocytes are then fused with an immortalized cell line using asuitable fusing agent, such as polyethylene glycol, to form a hybridomacell [Goding, Monoclonal Antibodies: Principles and Practice, AcademicPress, (1986) pp. 59-103]. Immortalized cell lines are usuallytransformed mammalian cells, particularly myeloma cells of rodent,bovine and human origin. Usually, rat or mouse myeloma cell lines areemployed. The hybridoma cells may be cultured in a suitable culturemedium that preferably contains one or more substances that inhibit thegrowth or survival of the unfused, immortalized cells. For example, ifthe parental cells lack the enzyme hypoxanthine guanine phosphoribosyltransferase (HGPRT or HPRT), the culture medium for the hybridomastypically will include hypoxanthine, aminopterin, and thymidine (“HATmedium”), which substances prevent the growth of HGPRT-deficient cells.

Preferred immortalized cell lines are those that fuse efficiently,support stable high level expression of antibody by the selectedantibody-producing cells, and are sensitive to a medium such as HATmedium. More preferred immortalized cell lines are murine myeloma lines,which can be obtained, for instance, from the Salk Institute CellDistribution Center, San Diego, Calif. and the American Type CultureCollection, Manassas, Va. Human myeloma and mouse-human heteromyelomacell lines also have been described for the production of humanmonoclonal antibodies [Kozbor, J. Immunol., 133:3001 (1984); Brodeur etal., Monoclonal Antibody Production Techniques and Applications, MarcelDekker, Inc., New York, (1987) pp. 51-63].

The culture medium in which the hybridoma cells are cultured can then beassayed for the presence of monoclonal antibodies directed against NGL-1receptor. Preferably, the binding specificity of monoclonal antibodiesproduced by the hybridoma cells is determined by immunoprecipitation orby an in vitro binding assay, such as radioimmunoassay (RIA) orenzyme-linked immunoabsorbent assay (ELISA). Such techniques and assaysare known in the art. The binding affinity of the monoclonal antibodycan, for example, be determined by the Scatchard analysis of Munson andPollard, Anal. Biochem., 107:220 (1980).

After the desired hybridoma cells are identified, the clones may besubcloned by limiting dilution procedures and grown by standard methods[Goding, supra]. Suitable culture media for this purpose include, forexample, Dulbecco's Modified Eagle's Medium and RPMI-1640 medium.Alternatively, the hybridoma cells may be grown in vivo as ascites in amammal.

The monoclonal antibodies secreted by the subclones may be isolated orpurified from the culture medium or ascites fluid by conventionalimmunoglobulin purification procedures such as, for example, proteinA-Sepharose, hydroxylapatite chromatography, gel electrophoresis,dialysis, or affinity chromatography.

The monoclonal antibodies may also be made by recombinant DNA methods,such as those described in U.S. Pat. No. 4,816,567. DNA encoding themonoclonal antibodies of the invention can be readily isolated andsequenced using conventional procedures (e.g., by using oligonucleotideprobes that are capable of binding specifically to genes encoding theheavy and light chains of murine antibodies). The hybridoma cells of theinvention serve as a preferred source of such DNA. Once isolated, theDNA may be placed into expression vectors, which are then transfectedinto host cells such as simian COS cells, Chinese hamster ovary (CHO)cells, or myeloma cells that do not otherwise produce immunoglobulinprotein, to obtain the synthesis of monoclonal antibodies in therecombinant host cells. The DNA also may be modified, for example, bysubstituting the coding sequence for human heavy and light chainconstant domains in place of the homologous murine sequences [U.S. Pat.No. 4,816,567; Morrison et al., supra] or by covalently joining to theimmunoglobulin coding sequence all or part of the coding sequence for anon-immunoglobulin polypeptide. Such a non-immunoglobulin polypeptidecan be substituted for the constant domains of an antibody of theinvention, or can be substituted for the variable domains of oneantigen-combining site of an antibody of the invention to create achimeric bivalent antibody.

The antibodies may be monovalent antibodies. Methods for preparingmonovalent antibodies are well known in the art. For example, one methodinvolves recombinant expression of immunoglobulin light chain andmodified heavy chain. The heavy chain is truncated generally at anypoint in the Fc region so as to prevent heavy chain crosslinking.Alternatively, the relevant cysteine residues are substituted withanother amino acid residue or are deleted so as to prevent crosslinking.In vitro methods are also suitable for preparing monovalent antibodies.

The anti-NGL-1 antibodies of the invention may further be humanizedantibodies or human antibodies. Humanized forms of non-human (e.g.,murine) antibodies are chimeric immunoglobulins, immunoglobulin chainsor fragments thereof (such as Fv, Fab, Fab′, F(ab′)₂ or otherantigen-binding subsequences of antibodies) which contain minimalsequence derived from non-human immunoglobulin. Humanized antibodiesinclude human immunoglobulins (recipient antibody) in which residuesfrom a complementary determining region (CDR) of the recipient arereplaced by residues from a CDR of a non-human species (donor antibody)such as mouse, rat or rabbit having the desired specificity, affinityand capacity. In some instances, Fv framework residues of the humanimmunoglobulin are replaced by corresponding non-human residues.Humanized antibodies may also comprise residues which are found neitherin the recipient antibody nor in the imported CDR or frameworksequences. In general, the humanized antibody will comprisesubstantially all of at least one, and typically two, variable domains,in which all or substantially all of the CDR regions correspond to thoseof a non-human immunoglobulin and all or substantially all of the FRregions are those of a human immunoglobulin consensus sequence. Thehumanized antibody optimally also will comprise at least a portion of animmunoglobulin constant region (Fc), typically that of a humanimmunoglobulin [Jones et al., Nature, 321:522-525 (1986); Riechmann etal., Nature, 332:323-329 (1988); and Presta, Curr. Op. Struct. Biol.,2:593-596 (1992)].

Methods for humanizing non-human antibodies are well known in the art.Generally, a humanized antibody has one or more amino acid residuesintroduced into it from a source which is non-human. These non-humanamino acid residues are often referred to as “import” residues, whichare typically taken from an “import” variable domain. Humanization canbe essentially performed following the method of Winter and co-workers[Jones et al., Nature, 321:522-525 (1986); Riechmann et al., Nature,332:323-327 (1988); Verhoeyen et al., Science, 239:1534-1536 (1988)], bysubstituting rodent CDRs or CDR sequences for the correspondingsequences of a human antibody. Accordingly, such “humanized” antibodiesare chimeric antibodies (U.S. Pat. No. 4,816,567), wherein substantiallyless than an intact human variable domain has been substituted by thecorresponding sequence from a non-human species. In practice, humanizedantibodies are typically human antibodies in which some CDR residues andpossibly some FR residues are substituted by residues from analogoussites in rodent antibodies.

Human antibodies can also be produced using various techniques known inthe art, including phage display libraries [Hoogenboom and Winter, J.Mol. Biol., 227:381 (1991); Marks et al., J. Mol. Biol., 222:581(1991)]. The techniques of Cole et al. and Boerner et al. are alsoavailable for the preparation of human monoclonal antibodies (Cole etal., Monoclonal Antibodies and Cancer Therapy, Alan R. Liss, p. 77(1985) and Boerner et al., J. Immunol., 147(1):86-95 (1991)]. Similarly,human antibodies can be made by introducing of human immunoglobulin lociinto transgenic animals, e.g., mice in which the endogenousimmunoglobulin genes have been partially or completely inactivated. Uponchallenge, human antibody production is observed, which closelyresembles that seen in humans in all respects, including generearrangement, assembly, and antibody repertoire. This approach isdescribed, for example, in U.S. Pat. Nos. 5,545,807; 5,545,806;5,569,825; 5,625,126; 5,633,425; 5,661,016, and in the followingscientific publications: Marks et al., Bio/Technology 10, 779-783(1992); Lonberg et al., Nature 368 856-859 (1994); Morrison, Nature 368,812-13 (1994); Fishwild et al, Nature Biotechnology 14, 845-51 (1996);Neuberger, Nature Biotechnology 14, 826 (1996); Lonberg and Huszar,Intern. Rev. Immunol. 13 65-93 (1995).

Mendez et al. (Nature Genetics 15: 146-156 (1997)) have further improvedthe technology and have generated a line of transgenic mice designatedas “Xenomouse II” that, when challenged with an antigen, generates highaffinity fully human antibodies. This was achieved by germ-lineintegration of megabase human heavy chain and light chain loci into micewith deletion into endogenous J_(H) segment as described above. TheXenomouse II harbors 1,020 kb of human heavy chain locus containingapproximately 66 V_(H) genes, complete D_(H) and J_(H) regions and threedifferent constant regions (μ, δ and χ), and also harbors 800 kb ofhuman κ locus containing 32 Vκ genes, Jκ segments and Cκ genes. Theantibodies produced in these mice closely resemble that seen in humansin all respects, including gene rearrangement, assembly, and repertoire.The human antibodies are preferentially expressed over endogenousantibodies due to deletion in endogenous J_(H) segment that preventsgene rearrangement in the murine locus.

Alternatively, the phage display technology (McCafferty et al., Nature348, 552-553 (1990)) can be used to produce human antibodies andantibody fragments in vitro, from immunoglobulin variable (V) domaingene repertoires from unimmunized donors. According to this technique,antibody V domain genes are cloned in-frame into either a major or minorcoat protein gene of a filamentous bacteriophage, such as M13 or fd, anddisplayed as functional antibody fragments on the surface of the phageparticle. Because the filamentous particle contains a single-strandedDNA copy of the phage genome, selections based on the functionalproperties of the antibody also result in selection of the gene encodingthe antibody exhibiting those properties. Thus, the phage mimics some ofthe properties of the B-cell. Phage display can be performed in avariety of formats; for their review see, e.g. Johnson, Kevin S, andChiswell, David J., Current Opinion in Structural Biology 3, 564-571(1993). Several sources of V-gene segments can be used for phagedisplay. Clackson et al., Nature 352 624-628 (1991) isolated a diversearray of anti-oxazolone antibodies from a small random combinatoriallibrary of V genes derived from the spleens of immunized mice. Arepertoire of V genes from unimmunized human donors can be constructedand antibodies to a diverse array of antigens (including self-antigens)can be isolated essentially following the techniques described by Markset al., J. Mol. Biol. 222, 581-597 (1991), or Griffith et al., EMBO J.12, 725-734 (1993). In a natural immune response, antibody genesaccumulate mutations at a high rate (somatic hypermutation). Some of thechanges introduced will confer higher affinity, and B cells displayinghigh-affinity surface immunoglobulin are preferentially replicated anddifferentiated during subsequent antigen challenge. This natural processcan be mimicked by employing the technique known as “chain shuffling”(Marks et al., Bio/Technol 10, 779-783 [1992]). In this method, theaffinity of “primary” human antibodies obtained by phage display can beimproved by sequentially replacing the heavy and light chain V regiongenes with repertoires of naturally occurring variants (repertoires) ofV domain genes obtained from unimmunized donors. This techniques allowsthe production of antibodies and antibody fragments with affinities inthe nM range. A strategy for making very large phage antibodyrepertoires has been described by Waterhouse et al., Nucl. Acids Res.21, 2265-2266 (1993).

Various techniques have been developed for the production of antibodyfragments. Traditionally, these fragments were derived via proteolyticdigestion of intact antibodies (see, e.g., Morimoto et al., J. Biochem.Biophys. Methods 24:107-117 (1992) and Brennan et al., Science 229:81(1985)). However, these fragments can now be produced directly byrecombinant host cells. For example, Fab′-SH fragments can be directlyrecovered from E. coli and chemically coupled to form F(ab′)₂ fragments(Carter et al., Bio/Technology 10:163-167 (1992)). In anotherembodiment, the F(ab′)₂ is formed using the leucine zipper GCN4 topromote assembly of the F(ab′)₂ molecule. According to another approach,Fv, Fab or F(ab′)₂ fragments can be isolated directly from recombinanthost cell culture. Other techniques for the production of antibodyfragments will be apparent to the skilled practitioner.

Heteroconjugate antibodies, composed of two covalently joinedantibodies, are also within the scope of the present invention. Suchantibodies have, for example, been proposed to target immune systemcells to unwanted cells (U.S. Pat. No. 4,676,980), and for treatment ofHIV infection (PCT application publication Nos. WO 91/00360 and WO92/200373). Heteroconjugate antibodies may be made using any convenientcross-linking methods, using well known, commercially availablecross-linking agents.

For further information concerning the production of monoclonalantibodies see also Goding, J. W., Monoclonal Antibodies: Principles andPractice, 3rd Edition, Academic Press, Inc., London, San Diego, 1996;Liddell and Weeks: Antibody Technology: A Comprehensive Overview, BiosScientific Publishers: Oxford, UK, 1995; Breitling and Dubel:Recombinant Antibodies, John Wiley & Sons, New York, 1999; and PhageDisplay: A Laboratory Manual, Barbas et al., editors, Cold SpringsHarbor Laboratory, Cold Spring Harbor, 2001.

The agonist antibodies of the invention may be selected, for example, byimmobilizing a Netrin-G1 receptor and then panning a library of humanscFv prepared as described above using the immobilized receptor to bindantibody. Griffiths et. al., EMBO-J, 1993, 12:725-734. The specificityand activity of specific clones can be assessed using known assays.After a first panning step, one obtains a library of phage containing aplurality of different single chain antibodies displayed on phage havingimproved binding to the receptor. Subsequent panning steps provideadditional libraries with higher binding affinities. When avidityeffects are a problem, monovalent phage display libraries may be used inwhich less than 20%, preferably less than 10%, and more preferably lessthan 1% of the phage display more than one copy of an antibody on thesurface of the phage. Monovalent display can be accomplished with theuse of phagemid and helper phage as described, for example, in Lowmanet. al., Methods: A Companion to Methods in Enzymology, 1991,3(3):205-216. A preferred phage is M13 and display is preferably as afusion protein with coat protein 3 as described in Lowman et. al.,supra. Other suitable phage include fl and fd filamentous phage. Fusionprotein display with other virus coat proteins is also known and may beused in this invention. See U.S. Pat. No. 5,223,409.

3. Target Diseases

Thalamocortical axons originate in dorsal thalamus, and projectventrally in diencephalon and then dorsolaterally in ventraltelencephalon to their target, the neocortex.

Netrin G1 and NGL-1 have been implicated in various neural diseases,including, without limitation, Alzheimer's disease, Parkinsons'sdisease, Huntington's chorea, amyotrophic lateral sclerosis (ALS),peripheral neuropathies, and other conditions characterized byneurodegeneration or loss of neurons. In addition, the composition maybe useful in treating damaged nerve cells, e.g., nerves damaged bymedical conditions such as strokes, diabetes mellitus, liver or kidneydysfunction or other endocrine/metabolic derangements, and the toxiceffect of chemotherapy or radiation used to treat cancer, HIV and AIDS.

Target diseases for treatment with NGL-1 or agonists thereof include,without limitation, benign or malignant brain tumors of any cellularorigin and of any location, optionally combined with surgical, chemicaland/or radiological treatment of such tumors; mechanical trauma of thenervous system (including the brain, spinal cord, nerve roots,peripheral ganglia and peripheral nerves) that impinges upon, ordisrupts the integrity of any axonal pathways, including thethalamocortical pathway; chemical intoxication of the nervous systemaffecting axons, such as mercury and lead; congenital or hereditaryabnormalities of the nervous system affecting axonal growth or function,such as, for example, Charcot-Marie-Tooth diseases; autoimmune diseasesattacking the axons of the central or peripheral nervous system, such asmultiple sclerosis and Gullian-Barre syndrome; viral, bacterial orparasitic infections of the nervous system with damage to the axons,such as acute or chronic meningitis, encephalitis or neuritis caused byherpes simplex virus, HIV, tuberculosis and/or leprosy.

4. Pharmaceutical Compositions

NGL-1, antibodies specifically binding an NGL-1 receptor, as well asother NGL-1 agonist molecules identified by the screening assaysdisclosed hereinbefore, can be administered for the treatment of variousdisorders, in particular the target diseases listed above, in the formof pharmaceutical compositions.

Where antibody fragments are used, the smallest inhibitory fragment thatspecifically binds to the binding domain of the target protein ispreferred. For example, based upon the variable-region sequences of anantibody, peptide molecules can be designed that retain the ability tobind the target protein sequence. Such peptides can be synthesizedchemically and/or produced by recombinant DNA technology. See, e.g.,Marasco et al., Proc. Natl. Acad. Sci. USA, 90: 7889-7893 (1993).

The active ingredients may also be entrapped in microcapsules prepared,for example, by coacervation techniques or by interfacialpolymerization, for example, hydroxymethylcellulose orgelatin-microcapsules and poly-(methylmethacylate) microcapsules,respectively, in colloidal drug delivery systems (for example,liposomes, albumin microspheres, microemulsions, nano-particles, andnanocapsules) or in macroemulsions. Such techniques are disclosed inRemington's Pharmaceutical Sciences, supra.

The formulations to be used for in vivo administration must be sterile.This is readily accomplished by filtration through sterile filtrationmembranes.

Sustained-release preparations may be prepared. Suitable examples ofsustained-release preparations include semipermeable matrices of solidhydrophobic polymers containing the antibody, which matrices are in theform of shaped articles, e.g., films, or microcapsules. Examples ofsustained-release matrices include polyesters, hydrogels (for example,poly(2-hydroxyethyl-methacrylate), or poly(vinylalcohol)), polylactides(U.S. Pat. No. 3,773,919), copolymers of L-glutamic acid and yethyl-L-glutamate, non-degradable ethylene-vinyl acetate, degradablelactic acid-glycolic acid copolymers such as the LUPRON DEPOT™(injectable microspheres composed of lactic acid-glycolic acid copolymerand leuprolide acetate), and poly-D-(−)-3-hydroxybutyric acid. Whilepolymers such as ethylene-vinyl acetate and lactic acid-glycolic acidenable release of molecules for over 100 days, certain hydrogels releaseproteins for shorter time periods. When encapsulated antibodies remainin the body for a long time, they may denature or aggregate as a resultof exposure to moisture at 37° C., resulting in a loss of biologicalactivity and possible changes in immunogenicity. Rational strategies canbe devised for stabilization depending on the mechanism involved. Forexample, if the aggregation mechanism is discovered to be intermolecularS—S bond formation through thio-disulfide interchange, stabilization maybe achieved by modifying sulhydryl residues, lyophilizing from acidicsolutions, controlling moisture content, using appropriate additives,and developing specific polymer matrix compositions.

The formulation herein may also contain more than one active compound asnecessary for the particular indication being treated, preferably thosewith complementary activities that do not adversely affect each other.Such molecules are suitably present in combination in amounts that areeffective for the purpose intended, or may be formulated separately, andadministered concurrently or consecutively, in any order.

For example, the NGL-1 agonists of the present invention may beadministered in combination with anti-neural agents and other activecompounds currently in use for the treatment of the target diseases andconditions discussed above.

The following example is offered for illustrative purposes only, and isnot intended to limit the scope of the present invention in any way.

All patent and literature references cited in the present specificationare hereby incorporated by reference in their entirety.

EXAMPLES

All cDNA plasmids and proteins used in the following examples weregenerated using standard molecular biological methods, or obtained fromcommercial vendors.

The soluble GFRα3 protein was kindly provided by D. Stone (RinatNeuroscience). Recombinant cMet-Fc fusion protein and recombinant6×His-tagged chick Netrin-1 protein were from R&D Systems.

Example 1 Specific and Direct Interaction between Netrin-G and NGL-1

The extracellular domain (ECD) of a group of 300 secreted ortransmembrane human proteins was fused with the constant region of humanimmunoglobulin G (IgG Fc). The ECD of selected cDNAs were fused in framewith the human IgG1 Fc region in the pRK5 vector for mammalianexpression or in pHIF (Pharmingen) for baculoviral production. Therecombinant human ECD-Fc and -his fusion proteins were produced by thebaculoviral expression system as described [Lee, J. et al. IL-17E, anovel proinflammatory ligand for the IL-17 receptor homolog IL-17Rh1. JBiol Chem 276, 1660-4. (2001)], or produced in HEK293 cells and purifiedby Ni-NTA column (Qiagen).

The resultant fusion proteins were expressed and used in binding assaysover a panel of ˜400 putative cell surface transmembrane or GPI-anchoredcell surface human proteins that were transiently expressed in COS7cells. Purified ECD-Fc fusion proteins (0.5 μg/ml) or the crudebaculovirus lysates were added to transiently transfected COS7 cells for1-2 hours at room temperature. After 3 rinses with cold phosphatebuffered saline (PBS), the bound protein was cross-linked to the cellsurface with 4% paraformaldehyde. The bound Fc fusion protein wasdetected by biotinylated anti-human IgGFc and R-phycoerythrin-conjugatedstreptavidin (Jackson Immunoresearch). Cell surface staining wasvisualized by fluorescent microscopy and images were taken with thePenguin 800CL digital camera system (Pixera).

One cDNA clone corresponding to the “c” splice isoform of humanNetrin-G1, which is structurally related to the Netrin family of axonguidance molecules [Nakashiba, T. et al. Netrin-G1: a novel glycosylphosphatidylinositol-linked mammalian netrin that is functionallydivergent from classical netrins. J. Neurosci. 20, 6540-6550. (2000)],when expressed in COS cells, was specifically labeled by the ECD of anovel transmembrane protein, named NGL-1. In fact, the human NGL-1 wasthe only protein that bound the human Netrin-G1 expressing COS cells inthis screen.

Although the NGL-1 and Netrin-G1 interaction appeared to be specificwithin the context of the screen, the classical Netrins and their knownreceptors, DCC and Unc5, were not included in the screen. Thus whetherNGL-1 also interacts with Netrin-1 and its receptors was determined.Using the same cell surface binding assay, NGL-1, Netrin-G1, or Netrin-1were expressed in COS7 cells (rows in FIG. 1 a-i) and their ability tobind the IgG Fe fusion protein of NGL-1, Netrin-G1, or DCC (columns inFIG. 1 a-i) was determined. The NGL-1-ECD IgG Fc fusion protein onlybound COS cells expressing Netrin-G1, but not cells that expressedNGL-1, Netrin-1 or DCC (FIG. 1 a-c and data not shown). Likewise, theNetrin-G1-Fc fusion protein bound only COS cells that expressed NGL-1,but not cells that expressed Netrin-G1, DCC or netrin (FIG. 1 d-f anddata not shown). Finally, DCC-Fc only bound to COS cells expressingNetrin-1, but not to cells expressing NGL-1, Netrin-G1 or DCC (FIG. 1g-I, and data not shown). These results indicate that the interactionbetween Netrin-G1 and NGL-1 is specific even within the Netrinsuperfamily and that Netrin-G1 and NGL-1 do not display homophilicinteractions.

It was noted that the labeling of Netrin-1-expressing-cell by DCC-Fc wasdiffuse (FIG. 1 i) due to Netrin-1 secretion and its affinity toheparin-like molecules on neighboring cell surface [Serafini, T. et al.The netrins define a family of axon outgrowth-promoting proteinshomologous to C. elegans UNC-6. Cell 78, 409-424. (1994)]. In contrast,the labeling of NGL-1 or Netrin-G1-expressing cells was fairly discrete(FIG. 1 b, d) despite the fact that they also exhibited affinities todextran and heparin-like molecules (by BIAcore, data not shown),suggesting that NGL-1 and Netrin-G1 are not released quantitatively fromthe surface of their respective producing cells.

The question remained whether Netrin-G1 and NGL-1 directly interact witheach other. Purified Netrin-G1 protein with a C-terminus His tag(“NetrinG-his”) was generated and was incubated with purified NGL-1-Fc.Recombinant ECD-Fc fusion proteins (5 nM) indicated at the top (FIG. 1j, lanes 3-7) were incubated with 5 nM of human Netrin-G1-his (firstrow), chicken Netrin-1-his (second row) or human HGF protein (third row)in a solution binding assay. The protein mixtures, each at 1-2 nM finalconcentration, were incubated at room temperature for 30 min, and then 1uL of the protein A beads (Pierce) was added to each mixture. The boundprotein A beads were then washed 3 times with the wash buffer bycentrifugation and resuspension. The wash buffer consists of 50 mM NaCl,10 mM TrisCl (pH7.5), 1 mM EDTA and 0.1% Triton X-100. Equal volume ofthe protein complexes were loaded and the blot was probed with HisProbe(“anti-His”) (Qiagen), anti-chicken Netrin-1 (R&D Systems) or ananti-human HGF monoclonal antibody (anti-HGF) (Genentech) as indicatedto the right in FIG. 1.

The NetrinG-his protein could be precipitated by NGL-1-Fc fusion protein(FIG. 1 j, first row) but not by DCC-Fc, Unc5h-Fc, or Met-Fc. Asexpected, DCC-Fc and Unc5h-Fc, but not NGL-1-Fc, could precipitateNetrin-1 (Netrin1-his, FIG. 1 j, second row). Also as expected, Met-Fcprecipitated its ligand the hepatocyte growth factor (HGF) (FIG. 1 j,third row).

Direct and specific binding of NGL-1-Fc and Netrin-G1-his (˜50 kDa) isshown in the first row. The Netrin1-his protein (75-85 kDa) bindsstrongly to DCC-Fc and Unc5-Fc (lanes 3-4, second row), extremely weaklywith NGL-1-Fc (lane 5, second row), and not at all to human cMet-Fc(lane 6). HGF binds cMet-Fc (lane 6, third row). Therefore, NGL-1 andNetrin-G1 interact directly without any requirement for additionalfactors.

Example 2 Cell Expression

To directly confirm that NGL-1 is membrane bound, NGL-1 wasepitope-tagged at the N-terminus immediately following the signalsequence and expressed in HEK293 cells. Although a full length NGL-1protein was detected in the total cell lysate, little or noepitope-tagged NGL-1 was detected in the medium. In contrast, secretedmyc-tagged Netrin-1 was readily detectable in the cell culture medium).Therefore, most if not all of the NGL-1 protein is retained on thesurface of the NGL-1 expressing cells.

Example 3 The Binding Affinity of NGL-1

The binding affinity of NGL-1 and Netrin-G1 was then evaluated by asolid phase binding assay. Crude NetrinG-his protein (1 μg/ml) wascoated in microtiter wells and non-specific binding sites saturated with0.5% BSA. Purified NGL-1-Fc protein was serially diluted and added tothe coated wells for 1 hour. Signals were detected with the biotinylatedanti-human IgGFc antibody, horseradish peroxidase-conjugated strepavidinand a chromogenic substrate TMB (Kirkgaard and Perry Lab). The reactionswere terminated with phosphoric acid and absorbance at 450 nm wasmeasured. Non-specific binding was determined in parallel by omittingthe microtiter well coating or by incubating the wells with theirrelevant cMet-Fc fusion protein, and dissociation constants weredetermined using saturation curves and Scatchard analysis (FIG. 2 a).NGL-1 and Netrin-G1 were found to interact with an apparent K_(d) of 1.6nM, a value that is of the same order of magnitude as the interactionbetween Netrin1-Fc and DCC (5.2 nM) [Keino-Masu, K. et al. Deleted inColorectal Cancer (DCC) encodes a netrin receptor. Cell 87, 175-185.(1996)].

Example 4 The Structure of NGL-1

Full length human NGL-1 and Netrin-G1 cDNA clones were initiallyisolated through screening cDNA libraries by virtue of the presence ofputative signal sequences.

FIG. 2 b shows a schematic diagram summarizing the predicted structureof the extracellular region of human NGL-1 protein. SS, signal sequence;LRR (leucine-rich repeat); NT, N-terminal domain of LRR; CT, C-terminaldomain of LRR; Ig (immunoglobulin domain).

The human NGL-1 cDNA encodes a putative type I transmembrane proteinwith an N-terminal signal sequence, a single transmembrane domain of 640amino acid residues and a predicted molecular weight of 71.9 kDa (FIG. 2b and FIG. 3). The ECD of NGL-1 consists of 9 leucine rich repeats (LRR,residues 77-291) with the flanking LRR N-terminal domain (LRR-NT,residues 46-75) and LRR C-terminal domain (LRR-CT, residues 301-352),followed by an immunoglobulin domain (Ig domain 367-428). This isfollowed by a TM (transmembrane region), and PDZ motif (potential PDZdomain-binding motif). The 92 amino acid cytoplasmic region does notcontain any obvious structural consensus sequence except that theC-terminus sequence “ETQI” is a potential PDZ-domain binding motif.

To determine which portion of the NGL-1 molecule mediates Netrin-G1binding, we generated various deletion constructs in the ECD of NGL-1and tested their ability to bind Netrin-G1 in both cell surface andsolution binding assays (FIG. 2 b). The results from both assays wereidentical and indicated that the LRR region, but not the Ig domain, ofNGL-1 is both necessary and sufficient for the interaction withNetrin-G1. The protein expression of all deletion constructs wasverified by western blot.

Example 5 Sequence Analysis

BLAST search identified additional human cDNAs that showed similarity tothe human NGL-1 sequence (FIG. 3). In particular, human NAG14 (GenBankAccession No. AF196976, SEQ ID NO: 2) is a cDNA down regulated in braintumors, and it is 51% identical, 89% similar to human NGL-1. A thirdcDNA, HSM (GenBank Accession No. HSM802162, SEQ ID NO: 3), appears to bea partial clone but the available sequence also showed 54% identity toNGL-1 and 50% identity to human NAG14. These cDNAs together define adistinct human LRR-containing transmembrane protein family.

The human NGL-1 clone was found to be identical to a cDNA clone in theGenBank. (Accession Number AB046800, SEQ ID NO: 1).

A mouse EST clone (GenBank accession no. AW210185, IMAGE Consortium, SEQID NO: 4) similar to human NGL-1 was identified by BLAST. The entiremouse EST clone was sequenced and found to be over 99.9% identical tothe human NGL-1 in the amino acid sequence, representing the mouseortholog of human NGL-1.

The partial mouse NAG14 homologue and the partial chick NGL-1 cDNA werecloned by degenerate RT-PCR using E15 mouse whole brain and E5-6 chicktelencephalon as the source of RNA respectively. Full length human DCC,Netrin-1 and unc5h3 clones were amplified from cDNA libraries.

By degenerate RT-PCR, we also cloned a partial cDNA of the chick NGL-1gene. The amino acid sequence alignment of the human NGL-1 and relatedhuman proteins encoded by EST cDNAs shown in FIGS. 3 and 4 shows thathuman and mouse NAG14 (SEQ ID NOs: 2 and 6) and another related EST, HSM(GenBank Accession Number HSM802162, SEQ ID NO: 3) are closely relatedto NGL-1. The identical amino acid residues are shaded in black and theconserved residues in gray. Underlined are the predicted structuraldomains and motifs. Therefore the NGL-1 gene is conserved among thevertebrate species.

The closest invertebrate gene similar to NGL-1 is the Drosophila kek3, ahypothetical gene predicted by the Drosophila Genome Project, but thesetwo proteins do not show any similarity outside the LRR region. In thisregard it is noteworthy that there is no Netrin-G1 homologue identifiedin either the Drosophila or C. elegans genome [Nakashiba, T. et al.Netrin-G1: a novel glycosyl phosphatidylinositol-linked mammalian netrinthat is functionally divergent from classical netrins. J. Neurosci. 20,6540-6550. (2000)]. Therefore, NGL-1 and Netrin-G1 are probably recentaddition to the genome during animal evolution.

Example 6 Distribution of NGL-1 mRNA and NGL-1-Binding Activity In Vivo

Next the distribution of NGL-1 and Netrin-G1 transcripts in a panel offetal and adult human RNA samples was examined (FIG. 5 a).

Adult and fetal human RNA panels were from Clontech, and the filterswere hybridized with ³²P-labeled human NGL-1 and Netrin-G1 probesaccording to the manufacturer's instructions. NGL-1 mRNA was detectedspecifically in the fetal and adult brain tissue as two bands of 3.5 and4.8 kb.

On the other hand, Netrin-G1 was highly expressed in the developing andadult thalamus (FIG. 5 a and previously published data [Nakashiba, T. etal. Netrin-G1: a novel glycosyl phosphatidylinositol-linked mammaliannetrin that is functionally divergent from classical netrins. J.Neurosci. 20, 6540-6550. (2000)]). This complementary pattern ofexpression raised the possibility that NGL-1-NetrinG1 interaction may beinvolved in the development and/or the mature functions of thethalamocortical axons.

To investigate the developmental expression of NGL-1, we turned to themouse embryos (FIG. 5 b, c). Mouse NGL-1 cDNA was used to preparedigoxinin-labeled riboprobe for in situ hybridization with frozensections of E14 CD1 mouse brain. Timed pregnant CD1 mice were fromHilltop and Charles River.

Crude NGL-1-Fc and Netrin-G1-Fc protein preps were added to freshlydissected whole mount E12-E18 mouse brain in the presence of 0.01%Tween-20 to enhance Fc protein penetration. Alternatively, freshlydissected brains were imbedded in 3% agarose in HBSS to prepare 200 μmVibratome sections, which were then incubated with the Fc-fusionproteins. After several gentle washes in PBS, the tissues were fixedwith 4% paraformaldehyde and the endogenous HRP activity was quenched by0.03% H₂O₂. Bound Fc fusion protein was detected by HRP-conjugatedanti-human IgGFc and DAB substrate (Sigma).

The mouse NGL-1 mRNA was highly expressed in the developing cerebralcortex (Ctx) and the striatum (Str) at E14 and the individualneocortical areas such as the frontal, parietal and occipital lobes.Putamen, amydala, hippocampus and medulla oblongata exhibited a moderatelevel of NGL-1 mRNA expression. Caudate nucleus and thalamus expressedNGL-1 at a low level, whereas other brain regions exhibited very weak orno expression of NGL-1. As expected, Netrin-G1 mRNA was highly expressedin the thalamus with very low or no expression in most of the othertissue or brain regions examined. The developing striatum has beenproposed to be an intermediate target and the cerebral cortex is thefinal target for TCAs [Metin, C. & Godement, P. The ganglionic eminencemay be an intermediate target for corticofugal and thalamocorticalaxons. J. Neurosci 16, 3219-3235. (1996)].

In further support of this hypothesis, we found that the E14 mouse TCAsspecifically bound NGL-1 (FIG. 5 d-g). The NGL-1-Fc-bound structuresfollowed the classical trajectory of TCAs from the dorsal thalamus (DT)through the ventral thalamus, the striatum into the cerebral cortex,although the mass of dorsal thalamus per se was not labeled under thesame experimental conditions (FIG. 5 e). This result showed that theendogenous NGL-1-binding activity is highly concentrated in the axons,not the cell bodies, of the thalamic projection neurons. In addition,NGL-1-Fc also labeled the lateral olfactory tracts (LOT) coursing overthe ventral surface of the E17 mouse forebrain in the stereotypical arcpattern (FIG. 5 h). Since Netrin-G1 mRNA is highly expressed in thedorsal thalamus, which projects the TCAs, and in the mitral cells of theolfactory bulb, which give rise to the axons in the LOT, the axonalNetrin-G1 protein is likely to be the predominant NGL-1 bindingactivity.

Since the human NGL-1 and NAG14 genes share high sequence similarity aswell as the same overall domain structures, we cloned a cDNA of themouse homologue of NAG14 and used a region that is divergent betweenNGL-1 and NAG14 as the probe for in situ hybridization. This mouse NAG14probe showed an identical pattern of expression to that of NGL-1 in theE14 forebrain.

Example 7 NGL-1 Promote Neurite Outgrowth of the Thalamic Neurons ViaNetrin-G1

To test whether NGL-1 and Netrin-G1 are involved in the development ofthalamic axons, we used the dissociated thalamic neuronal culture system

Dorsal thalamic neurons from embryonic day 13.5 (E13.5) mice weredissociated and grown on 96 well tissue culture plates pre-coated withdesignated substrates for ˜2 days. They were then labeled with the vitaldye 5-chloromethylfluororecein diacetate (CM-FDA, Molecular Probes) andthe neuronal marker anti-type III β-Tubulin (Chemicon). Viable neuronsbearing at least one neurite 2 times the cell body diameter were countedusing the TE300 inverted microscope (Nikon). Statistical analysis wasperformed using Instat software (GraphPad).

FIG. 6 a-g shows dissociated cultures of E13-14 mouse thalamic neuronsgrown on control bovine serum albumin (BSA) substrate (a, b) or on humanNGL-1 substrate (c-f). Some of the cultures were treated with PIPLC (e,f) for 20 min before the addition of soluble Netrin-G1 (f) or solubleGFRα3 (g). 44-48 hours after the culture, the neurons were stained forthe vital dye CM-FDA (a, c, e, f) or for the neuronal marker, anti-typeIII β-Tubulin (b, d). The great majority of live cells were positive forneuronal marker (90-95%). (g) The number of live neurons bearing neuitesin each culture condition was counted using the criterion that includesonly the cells positive for CM-FDA staining and with the longest neuriteat least twice the length of the widest cell body diameter.

When E13 mouse thalamic neurons were grown on a control substrate ofBovine Serum Albumin (BSA), few neurons extended neurites over a periodof 42-48 hours in culture (FIG. 6 a-b). By contrast, manyprocess-bearing thalamic neurons were observed when NGL-1 protein wasimmobilized as the substrate for neuronal culture (FIG. 6 c-d). Similarnumbers of live cells were observed with the live dye CM-FDA in thepresence or absence of NGL-1 substrate (223±22 and 238±17 cells per wellfor BSA and NGL-1 substrate respectively). This neurite outgrowthactivity, however, appeared to be dependent on GPI-anchored protein(s)present on the surface of the thalamic neurons as phosphatidylinositolphospholipase C (PIPLC) treatment dramatically reduced the number of theprocess-bearing neurons (FIG. 6 e). To test if the GPI-anchoredNetrin-G1 is an essential co-receptor mediating the NGL-1-inducedneurite outgrowth, we drew analogy to the GDNF receptor system in whicha high concentration of soluble GFRα component can partially restore thesignaling activity after PIPLC treatment¹⁵. Indeed a high concentrationof soluble Netrin-G1 protein (sNetrinG, added at 10-20 μg/mL), but notsoluble GFRα3 (at 15 μg/mL), was capable of partially restoring theNGL-1-stimulated neurite outgrowth in PIPLC treated thalamic neurons(FIG. 6 f-g, P<0.01, F=13.87, One-way ANOVA test). Netrin-G1 has noeffect on thalamic neurons grown on BSA or neurons grown on NGL-1without PIPLC treatment (FIG. 6 g). These results strongly suggestedthat the surface bound NGL-1 can promote neurite outgrowth of developingthalamic neurons and that such activity is at least partially dependentupon the GPI-anchored Netrin-G1 present on the thalamic neurons.

Example 8 Soluble NGL-1 Blocks the Growth of Thalamic Axons In Vivo

Finally the question was asked whether the interaction of NGL-1 andNetrin-G1 is required for the growth of TCAs in vivo. To this aim thechick embryos were used as an experimental system. The avian dorsalthalamus also gives rise to a prominent bundle of thalamofugal axonsthat project to the telencephalon and can be visualized with theanti-Axonin-1 antibody.

Fertilized Leghorn chicken eggs were from Charles River/SPAFAS. Startingfrom Hamburger-Hamilton stage 12 (embryonic day 1.5), 0.2-1 μg ofNGL-1-Fc or cMet-Fc fusion protein was injected daily into theprosencephlic vesicle of the chick neural tube. At stages 25-26 (day5.5), the brains were dissected and analyzed by whole mountimmunohistochemistry with anti-Axonin-1 antibody (Developmental StudyHybridoma Bank).

We injected the soluble NGL-1-Fc or Netrin-G1-Fc protein into the neuraltube of the chick embryos daily starting from HH stages 10-12 andanalyzed the thalamofugal axons at stages 25-26 with anti-Axoninantibody (FIG. 6 h-i). A substantial number of the NGL-1-Fc injectedembryos exhibited severe reduction in Axonin-1-positive thalamofugalaxons (n=21/67, or 31%) (FIG. 6 i-j) as compared with the embryosinjected with PBS or a control Met-Fc (n=2/42, or 5%) (FIG. 6 h). Thisdifference is statistically significant (P=0.0007, Fisher's exact test).On the other hand, Netrin-G1-Fc injected embryos still maintained anormal pattern of thalamofugal axons (not shown), consistent with theobservation that excess of soluble Netrin-G1 protein did not affectNGL-1-induced neurite outgrowth of the murine thalamic neurons (FIG. 6g).

These experiments further support the importance of the NGL-1/NetrinG1interaction in the development of thalamic axons.

While the present invention has been described with reference to whatare considered to be the specific embodiments, it is to be understoodthat the invention is not limited to such embodiments. To the contrary,the invention is intended to cover various modifications and equivalentsincluded within the spirit and scope of the appended claims.

1. A method of promoting axonal growth or regeneration comprisingdelivering to an injured neuron an effective amount of a polypeptidehaving at least about 80% sequence identity with amino acid residues44-352 of SEQ ID NO: 1 and comprising a transmembrane region, or anagonist thereof.
 2. The method of claim 1 wherein said polypeptide hasat least about 85% sequence identity with amino acid residues 44-352 ofSEQ ID NO:
 1. 3. The method of claim 1 wherein said polypeptide has atleast about 90% sequence identity with amino acid residues 44-352 of SEQID NO:
 1. 4. The method of claim 1 wherein said polypeptide has at leastabout 95% sequence identity with amino acid residues 44-352 of SEQ IDNO:
 1. 5. The method of claim 1 wherein said polypeptide has at leastabout 99% sequence identity with amino acid residues 44-352 of SEQ IDNO:
 1. 6. The method of claim 1 wherein said polypeptide furthercomprises a C-terminal PDZ domain-binding motif.
 7. The method of claim6 wherein said PDZ domain-binding motif has the sequence of VQETQI (SEQID NO: 7).
 8. The method of claim 1 wherein said polypeptide comprisesan extracellular domain (ECD) comprising nine leucine-rich repeats(LRRs).
 9. The method of claim 1 wherein said polypeptide furthercomprises an N-terminal signal sequence.
 10. The method of claim 1wherein said polypeptide comprises amino acids 44-352 of SEQ ID NO: 1.11. The method of claim 1 wherein said polypeptide comprises amino acids44-428 of SEQ ID NO:
 1. 12. The method of claim 1 wherein saidpolypeptide comprises amino acids 44-546 of SEQ ID NO:
 1. 13. The methodof claim 1 wherein said polypeptide is human NGL-1 (SEQ ID NO: 1), withor without the N-terminal signal sequence and with or without animmunoglobulin-like region.
 14. The method of claim 1 wherein saidpolypeptide is a non-human homologue of human NGL-1 (SEQ ID NO: 1), withor without an N-terminal signal sequence and with or without animmunoglobulin-like region.
 15. The method of claim 14 wherein saidnon-human homologue is mouse NGL-1 (SEQ ID NO: 4).
 16. The method ofclaim 14 wherein said non-human homologue is chicken NLG-1 (SEQ ID NO:5).
 17. The method of claim 1 wherein said agonist is an agonistantibody.
 18. The method of claim 1 wherein said agonist is an antibodyfragment.
 19. The method of claim 17 or 18 wherein said antibody orantibody fragment is chimeric, humanized or human.
 20. The method ofclaim 1 wherein said agonist is a peptide.
 21. A method for treating adisease or condition associated with the loss, loss of function ordysfunction of nerve cells comprising delivering to said nerve cells aneffective amount of a polypeptide having at least 80% sequence identitywith amino acid residues 44-352 of SEQ ID NO: 1 and comprising atransmembrane region, or an agonist thereof.
 22. The method of claim 21wherein said nerve cells are thalamic nerve cells.
 23. The method ofclaim 21 wherein said disease or condition is a neurodegenerativedisease.
 24. The method of claim 21 wherein said disease or condition ischaracterized by nerve cell injury.
 25. The method of claim 24 whereinsaid injury is due to mechanical trauma.
 26. The method of claim 24wherein said injury is associated with diabetes, stroke, liver or kidneydysfunction, other endocrine or metabolic derangements, chemotherapy orradiation, or chemical intoxication of the nervous system.
 27. Themethod of claim 21 wherein said disease or condition is associated withspinal cord injury.
 28. The method of claim 27 wherein said disease orcondition is allodynia or pain following spinal cord injury.
 29. Themethod of claim 21 wherein said disease or condition is associated withneural dysfunction.
 30. The method of claim 29 wherein said disease orcondition is selected from the group consisting of Alzheimer's disease,Parkinson's disease, Huntington's chorea, amylotrophic lateral sclerosis(ALS), and peripheral neuropathies.
 31. The method of claim 21 whereinsaid disease or condition is a congenital or hereditary abnormality. 32.The method of claim 31 wherein said congenital or hereditary abnormalityis a Charcot-Marie-Tooth disease.
 33. The method of claim 21 whereinsaid disease or condition is an autoimmune disease attacking axons ofthe central or peripheral nervous system.
 34. The method of claim 33wherein said autoimmune disease is multiple sclerosis or Gulliam-Barresyndrome.
 35. A method for treating a disease or condition associatedwith the loss, loss of function or dysfunction of nerve cells comprisingdelivering to said nerve cells an effective amount of a nucleic acidencoding a polypeptide having at least 80% sequence identity with aminoacid residues 44-352 of SEQ ID NO: 1 and comprising a transmembraneregion, or a peptide or polypeptide agonist thereof.
 36. The method ofclaim 35 wherein said nerve cells are thalamic nerve cells.